Recent developments in the pharmaceutical industry and in combinatorial chemistry have exponentially increased the number of potentially useful chemical compounds. It is desirable to characterize these compounds to identify their active components and/or establish processes for their synthesis.
One useful analytical process is chromatography, which encompasses a number of methods that are used for separating ions or molecules that are dissolved in or otherwise mixed into a solvent. Liquid chromatography (“LC”) is a physical method of separation wherein a liquid “mobile phase” (typically consisting of one or more solvents) carries a sample containing multiple constituents or species through a separation medium or “stationary phase.” Various types of mobile phases and stationary phases may be used. Stationary phase material typically includes a liquid-permeable medium such as packed granules (particulate material) disposed within a tube. The packed material contained by the tube or similar boundary is commonly referred to as a “separation column.” High pressure is often used to obtain a close-packed column with a minimal void between each particle, since better separation results (resolution) is typically obtained from more tightly packed columns. As an alternative to packed particulate material, porous monoliths or similar microporous matrices may be used. So-called “high performance liquid chromatography” (“HPLC”) refers to efficient LC separation methods that are usually performed at high operating pressures.
Typical interactions between stationary phases and solutes include adsorption, ion-exchange, partitioning, and size exclusion. Examples of types of stationary phases to support such interactions are solids, ionic groups on a resin, liquids on an inert solid support, and porous or semi-porous inert particles, respectively. Commonly employed base materials include silica, alumina, zirconium, and polymeric materials. A stationary phase material may act as a sieve to perform simple size exclusion chromatography, or the stationary phase may include functional groups (e.g., chemical groups) to perform separations based on other interaction types such as adsorption or ion exchange.
Mobile phase is forced through the stationary phase using means such as, for example, one or more pumps, gravity, voltage-driven electrokinetic flow, or other established means for generating a pressure differential. After sample is injected into the mobile phase (e.g., using a conventional loop valve), components of the sample migrate according to interactions with the stationary phase and specific components are retarded to varying degrees as they flow through the column. Individual sample components may reside for some time in the stationary phase (where their velocity is essentially zero) until conditions (e.g., a change in solvent concentration) permit a component to emerge from the column with the mobile phase. In other words, as a sample travels through voids or pores in the stationary phase, the sample may be separated into its constituent species due to the attraction of the species to the stationary phase. The time a particular constituent spends in the stationary phase relative to the fraction of time it spends in the mobile phase will determine its velocity through the column. Following separation in a column, the resulting eluate stream (consisting of mobile phase and sample) contains series of regions each having an elevated concentration of different components of the sample. These components can be detected using various techniques, including both flow-through and consumptive (destructive) techniques. Conventional flow-through detection technologies include spectrophotometric (e.g., UV-Vis), fluorimetric, refractive index, electrochemical, or radioactivity detection. Mass spectrometric analysis and nuclear magnetic resonance are examples of conventional consumptive detection technologies.
Due to the recognized utility of LC and the growing demand to analyze chemical entities, it would be desirable to increase the rate at which such entities can be isolated and characterized. Researchers have sought to provide parallel LC systems to perform multiple chromatographic separations simultaneously. Nonetheless, the ability to perform multiple parallel separations has been limited for a variety of reasons.
Conventional HPLC separation columns, which are tube-based, require porous frits positioned at both ends of the tube to retain the stationary phase material along with ferrules and nuts or other appropriate fasteners. One drawback of this type of separation column is that its assembly is complex and time-consuming. Another drawback of conventional tube-based separation columns is they interface with other system components through threaded fittings, which are not amenable to automated engagement and disengagement due to the difficulty of manipulating such fittings along with strict alignment tolerances. The need to periodically change tube-based columns with threaded fittings also means that sufficient space must be provided between each column to permit them to be accessed with appropriate tools. As a result, conventional multi-column LC systems offer little benefit in terms of simplicity or volumetric savings with the addition of each incremental separation column.
It would be desirable to provide high throughput systems for performing multiple LC separations in parallel while permitting multiple separation columns to be easily installed and operated within the system. It also would be desirable to provide microfluidic interfaces capable of maintaining fluid-tight seals at the high operating pressures typically associated with high performance liquid chromatography.
Another difficulty with integrating a large number of separation columns into a single system includes providing sufficient detection capability. In conventional chromatography systems, each column has at least one dedicated detector. High-sensitivity detectors such as photomultiplier tubes are typically both expensive and bulky, thus limiting the scalability of multi-column LC systems and rendering it difficult for them to include large numbers of separation columns. Additionally, for flow-through analyses such as optical analyses to yield useful results, an optical path must be transmissive of radiation of the desired frequency and the path should further contain a sufficient volume of analyte to provide an unambiguous signal. Thus, while microscale systems would appear to offer advantages in terms of packaging multiple columns into a limited volume, such systems may suffer from limited sensitivity.
Thus, needs exist for improved liquid chromatography systems and methods.